Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 March 2018 - 13 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized
EC Number:
291-707-5
EC Name:
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Cas Number:
90459-62-4
Molecular formula:
C24H55N3O6S
IUPAC Name:
bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate
Test material form:
solid
Remarks:
paste

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
sodium phenobarbitone and β-naphthoflavone induced rat liver S9 fraction
Test concentrations with justification for top dose:
0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 66 hours and 45 mins (plate incorporation), 65 hrs and 30 mins (preincubation assay)

NUMBER OF REPLICATIONS: 3



Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation

RANGE-FINDING/SCREENING STUDIES:
Based on the results of precipitation test, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate were selected for the initial cytotoxicity test.
The test item resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. The tester strain exposed with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, 1+ (extremely reduced lawn) for 0.7 mg/plate, 2+ (moderately reduced lawn) for 0.6 mg/plate and 3 + (Slightly reduced lawn) for 0.5 mg/plate when compared to vehicle control.
The test item was found to be cytotoxic up to the maximum concentration of 0.5 mg/plate tested when compared to the vehicle control.
On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for the main mutagenicity assay.


Any other information on results incl. tables

Solubility Test

The test item formed milky white suspension in distilled water at a concentration of 50 mg/mL.

 

Precipitation Test

The test item resulted in no precipitation at and up to 5 mg/plate.

 

Initial Cytotoxicity Test

Based on the results of precipitation test, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate were selected for the initial cytotoxicity test.

The test item resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. The tester strain exposed with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, 1+ (extremely reduced lawn) for 0.7 mg/plate, 2+ (moderately reduced lawn) for 0.6 mg/plate and 3 + (Slightly reduced lawn) for 0.5 mg/plate when compared to vehicle control.

The test item was found to be cytotoxic up to the maximum concentration of 0.5 mg/plate tested when compared to the vehicle control.

On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for the main mutagenicity assay.

 

TABLE 1.            SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUM TA100TESTER STRAIN

Test Item Concentration (mg/plate)

No. of Revertants/plate

With S9

Without S9

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

Vehicle Control

94

90

98

94

4.0

4+

99

95

92

95

3.5

4+

0.5

53

58

61

57

4.0

3+

50

55

52

52

2.5

3+

0.6

32

39

30

34

4.7

2+

33

36

29

33

3.5

2+

0.7

15

18

16

16

1.5

1+

17

19

15

17

2.0

1+

0.8

0

0

0

0

0.0

0

0

0

0

0

0.0

0

0.9

0

0

0

0

0.0

0

0

0

0

0

0.0

0

1

0

0

0

0

0.0

0

0

0

0

0

0.0

0

2

0

0

0

0

0.0

0

0

0

0

0

0.0

0

3

0

0

0

0

0.0

0

0

0

0

0

0.0

0

4

0

0

0

0

0.0

0

0

0

0

0

0.0

0

5

0

0

0

0

0.0

0

0

0

0

0

0.0

0

Values of Revertants are in Mean±SD

Lawn intensity:

4+= Thick lawn: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.    

3+=Slightly thin lawn: Distinguished by a noticeable thinning (Slightly reduced) of the background lawn compared to vehicle control plates.

2+=Thin lawn: Distinguished by a marked thinning (Moderately reduced) of the background lawn compared to solvent/vehicle control plates.

1+=Very thin lawn: Distinguished by an extreme thinning(Extremely reduced)of the background lawn compared to solvent/vehicle control plates.

0=No lawn: Distinguished by a complete lack of any background lawn compared to solvent/vehicle control plates.

 

.    

 

TABLE 2.            SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I

          Plate Incorporation Method

                                                                                                                                         

Treatment

Test Concentration  (mg/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

Salmonella typhimurium

TA 98

TA

100

TA 102

TA 1535

TA 1537

TA 98

TA 100

TA 102

TA 1535

TA 1537

Vehicle Control

100 µL of Distilled water

Mean

29.0

110.0

264.3

23.7

10.0

27.3

114.3

264.0

22.3

10.0

±SD

3.6

5.6

12.9

2.5

3.6

3.2

22.0

7.9

3.1

2.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Leomin KP

0.005

Mean

29.0

101.7

265.0

23.3

11.0

27.0

113.3

262.3

22.0

8.7

±SD

4.0

4.7

11.4

2.5

1.0

3.0

21.1

5.5

2.0

3.8

Fold Increase

1.0

0.9

1.0

1.0

1.1

1.0

1.0

1.0

1.0

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.016

Mean

28.0

111.0

256.3

22.0

9.0

25.7

105.7

253.7

22.0

9.3

±SD

2.0

10.6

8.1

3.0

2.6

3.5

8.1

6.4

3.6

0.6

Fold Increase

1.0

1.0

1.0

0.9

0.9

0.9

0.9

1.0

1.0

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.05

Mean

28.0

108.0

264.0

21.7

9.3

26.7

101.0

264.0

21.3

8.3

±SD

4.0

6.0

11.5

3.2

2.1

2.5

10.6

5.0

2.5

3.2

Fold Increase

1.0

1.0

1.0

0.9

0.9

1.0

0.9

1.0

1.0

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.16

Mean

26.7

98.7

256.3

21.3

8.0

26.3

95.0

254.7

20.0

7.7

±SD

4.9

8.0

11.2

2.5

1.0

3.2

7.2

4.7

3.0

1.5

Fold Increase

0.9

0.9

1.0

0.9

0.8

1.0

0.8

1.0

0.9

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.5

Mean

17.0

57.7

208.7

16.7

5.3

15.7

57.3

207.7

13.7

4.0

±SD

2.6

4.0

7.5

0.6

3.2

1.5

5.0

6.7

1.5

1.0

Fold Increase

0.6

0.5

0.8

0.7

0.5

0.6

0.5

0.8

0.6

0.4

Lawn Intensity

3+

3+

3+

3+

3+

3+

3+

3+

3+

3+

Positive Control

100 µL of respective Positive Control

Mean

348.7

405.7

606.0

125.7

105.0

343.3

401.0

609.0

119.0

100.3

±SD

15.7

15.3

7.0

8.5

4.6

18.4

10.5

8.2

14.9

4.2

Fold Increase

12.0

3.7

2.3

5.3

10.5

12.6

3.5

2.3

5.3

10.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102: 0.5 µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.5 mg/plate under the test conditions.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD Guideline 471, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 ofS. typhimurium were exposed toOctadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternizedat concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test in the presence and absence of mammalian metabolic activation (sodium phenobarbitone and β-naphthoflavone induced rat liver S9 fraction). Distilled water was used as vehicle. The test was performed as plate incorporation and pre-incubation assay.

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was tested uo to cytotoxic concentrations. The positive controls induced the appropriate responses.

There was noevidenceof induced mutant colonies over background.